Flaviviruses including dengue, West Nile, yellow fever, and Japanese encephalitis viruses, pose significant threats as emerging diseases and potential bioterror agents. Despite the considerable impact of flavivirus infection on world-wide health, no antiviral therapies are available, and existing flavivirus vaccines are of limited utility. The long-term goal of this project is to develop antiviral therapeutics based on structural and biochemical information regarding the flavivirus replicase complex. Since replicase complexes are absolutely required for virus replication, knowledge of their structures and mechanisms should identify potential targets for therapeutics and suggest strategies for intervention against threats from these infectious diseases. The flavivirus replicase complex, consisting of a virally-encoded RNA polymerase and helicase as well as other viral and unidentified cellular proteins, is responsible for copying the viral genome. During replication, 5'-RNA capping of the nascent strand occurs along with RNA synthesis. The viral NS5 polymerase has both RNA-dependent RNA polymerase (RdRp) and 5'-RNA methyltransferase (MTase) activities within a single polypeptide, indicating that the RNA synthesis and capping processes may be coupled. Little is known as to whether or how the two activities are coordinated during replication, largely due to a lack of detailed structural information about the full-length polymerase. In Aim 1, the interactions of the RdRp and MTase domains within dengue virus NS5 and the conformational changes that occur therein during the polymerase and methylation reactions, will be determined by small-angle X-ray scattering in solution (Aim 1a). The resulting NS5 model will be tested in recombinant protein and in the infectious virus using site-directed mutagenesis (Aim 1b). In Aim 2, interactions of the full-length NS5 polymerase with RNA will be investigated. A 'dual' substrate that can bind simultaneously to both the RdRp and MTase domains will be designed and tested for its binding to the full- length NS5. In Aim 3, the X-ray crystal structures of the full-length dengue NS5 polymerase and its RNA complexes at different steps along the catalytic pathway will be determined. The combined structural, biochemical, and virological studies will help elucidate the mechanisms for RNA synthesis and its potential coordination with capping reactions in the NS5 polymerase.